Medicament comprising HGF gene

ABSTRACT

The present invention relates to a medicament comprising a HGF gene. The medicament of the present invention may be topically applied to the target organs so that the effects can be selectively exhibited, resulting in minimizing the side effects of HGF. &lt;IMAGE&gt;

TECHNICAL FIELD TO WHICH THE INVENTION PERTAINS

[0001] The present invention relates to a medicament for use in genetherapy and the like. More particularly, the present invention relatesto a medicament comprising a hepatocyte growth factor (HGF) gene as wellas a liposome containing the HGF gene.

PRIOR ART

[0002] HGF is a physiologically active peptide that exhibits diversepharmacological activities. The pharmacological activities of HGF aredescribed in, e.g., JIKKEN-IGAKU (Experimental Medicine), Vol. 10, No. 3(extra issue), 330-339 (1992). In view of its pharmacologicalactivities, HGF is expected to be useful as: a drug for the treatment ofcirrhosis of the liver or of renal diseases; epithelial cell growthaccelerators; anticancer agents; agents for the prevention of sideeffects in cancer therapy; agents for the treatment of lung disorders,gastrointestinal damages or cranial nerve disorders; agents for theprevention of side effects in immunosuppression; collagen degradationaccelerators; agents for the treatment of cartilage disorders, arterialdiseases, pulmonary fibrosis, hepatic diseases, blood coagulopathy,plasma hypoproteinosis or wounds; agents for the improvement of nervousdisorders; hematopoietic stem cell potentiators; and hair growthpromoters (Japanese Patent KOKAI (Laid-Open) Nos. 4-18028 and 4-49246,EP 492614, Japanese Patent KOKAI (Laid-Open) No. 6-25010, WO 93/8821,Japanese Patent KOKAI (Laid-Open) Nos. 6-172207, 7-89869 and 640934, WO94/2165, Japanese Patent KOKAI (Laid-Open) Nos. 6-40935, 6-56692 and741429, WO 93/3061, Japanese Patent KOKAI (Laid-Open) No. 5-213721,etc.).

[0003] As to gene therapy, extensive studies and investigations recentlyhave been made all over the world for treatment of adenosine deaminasedeficiency, AIDS, cancer, pustulous fibrosis or hemophilia, etc.

[0004] However, gene therapy using HGF genes is unknown yet. It is evenunclear if such gene therapy will be effective.

PROBLEMS TO BE SOLVED BY THE INVENTION

[0005] HGF is known to be one of the drugs that have a short half lifein blood. As a result, persistent topical administration for HGF wouldbe desirable.

[0006] In view of the diverse pharmacological activities of HGF, HGF isexpected to be developed as a drug having extensive applications tovarious diseases. On the other hand, when HGF is systemicallyadministered, side effects might be caused due to the diversepharmacological activities of HGF. In addition, when HGF itself isintravenously administered, HGF has the drawback that a considerableamount of HGF is retained in the liver, resulting in reduction of theamount of HGF that reachs the target organ.

MEANS FOR SOLVING THE PROBLEMS

[0007] The present invention has been made to solve the foregoingproblems. In summary, the present invention relates to:

[0008] (1) a medicament comprising a HGF gene;

[0009] (2) a liposome containing the HGF gene;

[0010] (3) a liposome according to (2), which is a membrane fusionliposome fused to Sendai virus;

[0011] (4) a medicament comprising the liposome according to (2) or (3);

[0012] (5) a medicament according to (1) or (4), for use in thetreatment of arterial disorders; and,

[0013] (6) a medicament according to (1) or (4), for use in thetreatment of cartilage injuries.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 shows expression of HGF in rat coronary endothelial cellssensitized with the hemagglutinating virus of Japan (HVJ)-liposome-DNAin Test Example 1.

[0015] In FIG. 2, the (line) graph shows cell growth rate in thepresence or absence of HGF from the HVJ-liposome-cont-sensitizedendothelial cells in Test Example 2, wherein “DSF” designates a group ofendothelial cells sensitized with HVJ-liposome-cont and “HGF” designatesa group of endothelial cells incubated in the presence of recombinanthuman HGF at a predetermined concentration. The bar in FIG. 2 shows thecell growth rate of the HVJ-liposome-DNA-sensitized endothelial cells inTest Example 2, wherein “DSF” designates a group of endothelial cellssensitized with HVJ-liposome-cont and “HGF vector” designates a group ofendothelial cells sensitized with HVJ-liposome-DNA.

[0016]FIG. 3 shows the cell growth rate of endothelial cells sensitizedwith HVJ-liposome-DNA in the presence or absence of anti-HGF antibody inTest Example 2, wherein “control” represents a group ofHVJ-liposome-cont-sensitized endothelial cells incubated in the presenceof IgG control; “HGF” represents a group of HVJ-liposome-DNA-sensitizedendothelial cells incubated in the presence of IgG control; and “HGFab”represents a group of HVJ-liposome-DNA-sensitized endothelial cellsincubated in the presence of rabbit anti-human HGF antibody. The cellgrowth rate (%) is expressed in terms of relative % when the growth ratein the control group is set as 100.

[0017]FIG. 4 is a graph showing the cell growth effect of culturesupernatant from HVJ-liposome-DNA-sensitized rat vascular smooth musclecells (hereinafter often abbreviated as VSMCs) on rat coronaryendothelial cells in Test Example 3, wherein “control” designates agroup to which was added the culture supernatant from theHVJ-liposome-cont-sensitized rat VSMCs, and “HGF” designates a group towhich was added culture supernatant from the HVJ-liposome-DNA-sensitizedrat VSMCs.

[0018]FIG. 5 is a graph showing the results from Test Example 3 in whichthe concentration of HGF in the supernatant from incubatedHVJ-liposome-DNA-sensitized rat VSMCs was determined using anti-humanHGF antibody. In the figure, “no-treatment” represents a group to whichwas added culture supernatant from non-sensitized VSMCs; “control”represents a group to which was added supernatant from the incubatedHVJ-liposome-cont-sensitized rat VSMCs; and “HGF” represents a group towhich was added supernatant from the incubatedHVJ-liposome-DNA-sensitized rat VSMCs.

[0019]FIG. 6 is a graph showing the results from Test Example 3 in whichthe concentration of HGF in the supernatant from the incubatedHVJ-liposome-DNA-sensitized rat VSMCs was determined using anti-rat HGFantibody. In the figure, “no-treatment” represents a group treated withthe culture supernatant of non-sensitized VSMCs; “control” designates agroup treated with the supernatant from the incubatedHVJ-liposome-cont-sensitized rat VSMCs; and “HGF” designates a grouptreated with the supernatant from the incubatedHVJ-liposome-DNA-sensitized rat VSMCs.

[0020]FIG. 7 is a graph showing the cell growth effect of thesupernatant from incubated HVJ-liposome-DNA-sensitized rat coronaryendothelial cells on rat coronary endothelial cells in Test Example 4,wherein A, B and C designate, respectively, a group to which was addedsupernatant from incubated HVJ-liposome-DNA-sensitized rat coronaryendothelial cells, a group to which was added supernatant from incubatedHVJ-liposome-cont-sensitized rat coronary endothelial cells, and a groupof no-treatment animals.

[0021]FIG. 8 shows the cell growth effect of HVJ-liposome-DNA-sensitizedrat coronary endothelial cells on rat coronary endothelial cells in thepresence of an anti-HGF antibody in Test Example 4. In the figure, Arepresents a group to which was added supernatant from the incubatedHVJ-liposome-DNA-sensitized rat coronary endothelial cells; B representsa group to which was added supernatant from the incubatedHVJ-liposome-cont-sensitized rat coronary endothelial cells; Crepresents a group to which was added supernatant from the incubatedHVJ-liposome-DNA-sensitized rat coronary endothelial cells and whereinanti-HGF antibody was added to the supernatant; and D represents a groupto which was added supernatant from the incubatedHVJ-liposome-DNA-sensitized rat coronary endothelial cells and whereincontrol antibody was added to the supernatant.

[0022]FIG. 9 is a drawing showing the cell growth of endothelial cellsin Test Example 5 when HVJ-liposome-DNA-sensitized human VSMCs wereco-incubated with non-sensitized human endothelial cells. In the figure,“control” represents a group of human endothelial cells co-incubatedwith HVJ-liposome-cont-sensitized VSMCs, and “HGF” represents a group ofhuman endothelial cells co-incubated with the supernatant from theincubated HVJ-liposome-DNA-sensitized VSMCs.

[0023]FIG. 10 indicates the cell growth of endothelial cells in TestExample 6 when HVJ-liposome-DNA-sensitized rat VSMCs were co-incubatedwith non-sensitized rat coronary endothelial cells. In the figure,“control” represents a group of human endothelial cells co-incubatedwith the HVJ-liposome-cont-sensitized VSMCs, and “HGF” represents agroup of human endothelial cells co-incubated with the culturesupernatant from the HVJ-liposome-DNA-sensitized VSMCs.

[0024]FIG. 11 shows an increase in the number of minute blood vessels inrat heart muscle directly injected with HVJ-liposome-DNA in Test Example8, wherein “HGF” denotes the number of minute blood vessels in rat heartmuscle directly injected with HVJ-liposome-DNA, and “control” denotesthe number of minute blood vessels in rat heart muscle directly injectedwith HVJ-liposome-cont.

[0025]FIG. 12 is a drawing showing that 3 weeks after administration ofHVJ-liposome-DNA into the joint, development of cartilage-like cells wasnoted in Test Example 9, as evidenced by synthesis of ToluidineBlue-stained proteoglycan.

[0026]FIG. 13 is a drawing showing that 4 weeks after administration ofHVJ-liposome-DNA into the joint, development of cartilage-like cells wasnoted in Test Example 9, as evidenced by the synthesis of ToluidineBlue-stained proteoglycan.

[0027]FIG. 14 is a drawing showing that even 4 weeks afteradministration of HVJ-liposome-DNA (TGF-β) prepared in ComparativeExample 2 into the joint, such development of cartilage-like cells asevidenced by the fact that synthesis of Toluidine Blue-stainedproteoglycan was not observed in Test Example 9.

[0028]FIG. 15 is a drawing showing that even 4 weeks afteradministration of HVJ-liposome-cont prepared in Comparative Example 1into the joint, no such development of cartilage-like cells as evidencedby synthesis of Toluidine Blue-stained proteoglycan was observed in TestExample 9.

BEST MODE FOR CARRYING OUT THE INVENTION

[0029] The “HGF gene” employed in the present invention means a genecapable of expressing HGF. Thus, so long as the polypeptide expressedhas substantially the same effect as that of HGF, the HGF gene may havea partial deletion, substitution or insertion of the nucleotidesequence, or may have other nucleotide sequences ligated therewith atthe 5′-terminus and/or 3′-terminus thereof. Typical examples of such HGFgenes include HGF genes as described in Nature, 342, 440 (1989),Japanese Patent KOKAI (Laid-Open) No. 5-111383, Biohem. Biophys. Res.Commun., 163, 967 (1989), etc. These genes may be used in the presentinvention.

[0030] The HGF gene is incorporated into an appropriate vector and theHGF gene-bearing vector is provided for use. For example, the HGF genemay be used in the form of a viral vector having the HGF gene asdescribed hereinafter, or in the form of an appropriate expressionvector having the HGF gene.

[0031] The “pharmaceutical composition” used in the present inventionmeans a medicament for the treatment or prevention of human diseases,which is attributed to the pharmacological activities of HGF. Forexample, exemplified are medicaments for the treatment or prevention ofthe diseases given hereinabove.

[0032] According to the present invention, the HGF gene is introducedinto cells wherein HGF is expressed in those cells to exhibit thepharmacological actions. Thus, the medicament of the present inventionis effectively applicable to the diseases for which HGF itself iseffective.

[0033] Where the HGF gene is introduced into, e.g., cells, the growth ofvascular endothelial cells is accelerated, while undesired growth ofvascular smooth muscle cells is not accelerated, as demonstrated in theExamples hereinafter. Moreover, as demonstrated in the Exampleshereinafter, where the HGF gene is introduced into the heart in in vivoanimal tests using rats, angiogenesis is observed. Therefore, the HGFgene is effective for the treatment and prevention of arterialdisorders, in particular, various diseases caused by a disturbance whichmainly involves abnormal proliferation of vascular smooth muscle cells(e.g., restenosis after percutaneous transluminal coronary angioplasty(PTCA), arteriosclerosis, insufficiency of peripheral circulation,etc.), and for the treatment and prevention of diseases such asmyocardial infarction, myocardia, peripheral angiostenosis, cardiacinsufficiency, etc. HGF itself is also useful for the treatment andprevention of the diseases as described above, since HGF promotes theproliferation of vascular endothelial cells but does not promote thegrowth of vascular smooth muscle cells. The pharmacological effects ofthe HGF gene are attributed to those of HGF itself.

[0034] As demonstrated in the Examples hereinafter, introduction of theHGF gene into the joint results in promoting repair of articularcartilage cells to thereby promote the proliferation ofproteoglycan-synthesizing cells. Therefore, the HGF gene is effectivefor the prevention and treatment of various cartilage injuries such asosteogenetic abnormality, arthritis deformans, discopathy deformans,fracture repair and restoration insufficiency, trauma caused by sports,key puncher's disease, etc. HGF itself is useful for the treatment andprevention of the diseases described above, since HGF promotes repairand growth of cartilage cells. The effects of the HGF gene are based onthose of HGF itself.

[0035] A “liposome” is a closed vesicle of lipid bilayer encapsulatingan aqueous compartment. It is known that the lipid bilayer membranestructure is extremely similar to biological membranes. To prepare theliposomes of the present invention, phospholipids are employed. Typicalexamples of phospholipids are phosphatidylcholines such as lecithin,lysolecithin, etc.; acidic phospholipids such as phosphatidylserine,phosphatidylglycerol, phosphatidylinositol, phosphatidylic acid, etc.;or phospholipids obtained by replacing an acyl group(s) of these acidicphospholipids with lauroyl, myristoyl, oleoyl, etc.; andsphingo-phospholipids such as phosphatidylethanolamine, sphingomyelin,etc. Neutral lipids such as cholesterol may also be added to thesephospholipids. The liposomes may be prepared, in a conventional manner,from naturally occurring materials such as lipids in normal cellmembranes. The liposomes containing the HGF gene of the presentinvention may be prepared, for example, by suspending a thin layer ofpurified phospholipids in a solution containing the HGF gene and thentreating the suspension in a conventional manner, such as byultrasonication.

[0036] The liposomes containing the HGF gene of the present inventionmay be appropriately fused to viruses, etc. to form membrane fusionliposomes. In this case, it is preferred to inactivate viruses, e.g.,through ultraviolet irradiation, etc. A particularly preferred exampleof the membrane fusion liposome is a membrane fusion liposome fused withSendai virus (hemagglutinating virus of Japan: HVJ). The membrane fusionliposome may be produced by the methods described in NIKKEI Science,April, 1994, pages 32-38; J. Biol. Chem., 266 (6), 3361-3364 (1991),etc. In more detail, the HVJ-fused liposome (HVJ-liposome) may beprepared, e.g., by mixing purified HVJ inactivated by ultravioletirradiation, etc. with a liposome suspension containing the HGF genevector, gently agitating the mixture and then removing unbound HVJ bysucrose density gradient centrifugation. The liposomes may be bound tosubstances having an affinity to target cells, thereby enhancing theefficiency of gene introduction into the target cells. Examples ofsubstances having an affinity for target cells include ligands such asan antibody, a receptor, etc.

[0037] For introduction of the HGF gene into cells, conventional methodsare employed, which are roughly classified into introduction via viralvectors and other strategies (NIKKEI Science, April, 1994, pages 2045;GEKKAN YAKUJI, 36 (1), 2348 (1994) and references cited therein). Bothtypes of methods can be used for preparation of the medicament of thepresent invention.

[0038] The former type of method using viral vectors comprises the stepof incorporating the HGF gene into, e.g., a retrovirus, an adenovirus,an adeno-related virus, a herpes virus, a vaccinia virus, a poliovirus,a sindbis virus or other RNA virus. Of these viruses, a retrovirus, anadenovirus and an adeno-related virus are particularly preferablyemployed for introduction.

[0039] Examples of the other methods include the liposome method,lipofectin method, microinjection method, calcium phosphate method,electroporation method. Of these methods, particularly preferred is theliposome method.

[0040] For practical use of the HGF gene as a medicament, it isadvantageous to introduce the HGF gene directly into the body (in vivomethod). Alternatively, certain cells are collected from the human, theHGF gene is then introduced into the cells outside the body, and cellshaving the HGF gene introduced therein are returned to the body (ex vivomethod). These methods are described in NIKKEI Science, April, 1994,pages 20-45; GEKKAN-YAKUJI, 36 (1), 23-48 (1994) and references citedtherein. Any of these methods is suitable, depending upon the disease tobe treated, target organs, etc. and may be applied to the medicamentcompositions of the present invention.

[0041] The in vivo method is less costly, less laborious and thereforemore convenient than the ex vivo method, but the latter method providesa higher efficiency of introduction of the HGF gene into cells.

[0042] Where the medicament of the present invention is administered bythe in vivo method, the medicament may be administered through any routeappropriate for diseases to be treated, such as via target organs, etc.The medicament may be administered intravenously, intraarterially,subcutaneously, intramuscularly, etc., or directly to the objectiveorgan of the disease, e.g., kidney, liver, lung, brain, nerve, etc.Direct administration to the objective site can treat the target organselectively. For example, in gene therapy using a gene for restenosisafter PTCA, the composition may be administered intraarterially(JIKKEN-IGAKU, 12 (extra issue 15), 1298-1933 (1994). Preferably, themedicament of the present invention is applied at the tip of a balloonused for PTCA and the tip is rubbed against a blood vessel, whereby themedicament may be introduced directly into vascular endothelial cellsand vascular smooth muscle cells.

[0043] Where the ex vivo method as described above is used to introducethe HGF gene, human cells (e.g., lymphocytes or hematopoietic stemcells) are harvested in a conventional manner, and the harvested cellsare sensitized with the medicament of the present invention for geneintroduction. Thereafter the HGF-producing cells are inserted back intothe human.

[0044] Where the medicament is administered by the in vivo method, themedicament may take various preparation forms, including the form of aliquid preparation. In general, the medicament may be preferablyprepared as an injection comprising the HGF gene as an activeingredient. If necessary and desired, conventional carriers may be addedto the composition. The injection may be prepared in a conventionalmanner, e.g., by dissolving the HGF gene in an appropriate solvent(e.g., sterilized water, a buffered solution, a physiological salinesolution, etc.), filtering the solution through a filter, etc. forsterilization, filling up a sterile container with the solution. Themedicament may be prepared using the HGF gene-containing viral vector,instead of the HGF gene itself. Where the liposomes containing the HGFgene embedded therein (or HVJ-liposomes) are employed, the medicamentmay be in the form of liposome preparations such as a suspension, afrozen preparation, a centrifugally concentrated frozen preparation,etc.

[0045] The content of the HGF gene in the medicament may beappropriately varied depending upon diseases to be treated, targetorgans, patients' ages or body weights, etc. However, it is appropriateto administer in a dose of 0.0001 mg to 100 mg, preferably 0.001 mg to10 mg when calculated as the HGF gene. The dose may be divided intoseveral days or a few months.

EXAMPLES

[0046] Hereinafter the present invention will be described in moredetail with reference to the examples but is not deemed to be limitedthereto. Materials and methods used in the following examples areoutlined below.

[0047] Materials and Methods

[0048] (1) HGF Expression Vector

[0049] The HGF expression vector was prepared by inserting human HGFcDNA (2.2 kb, Biochem. Biophys. Res. Commun., 172, 321-327 (1990);Japanese Patent KOKAI (Laid-Open) No. 5-111383) between the EcoRI andNotI sites of a pUC-SRα expression vector (FEBS, 333, 61-66 (1993)). Inthis plasmid vector, transcription of HGF cDNA is regulated by the SRapromoter (Nature, 342, 440-443 (1989)).

[0050] (2) Cell Culture

[0051] Rat coronary endothelial cells were isolated from enzymaticallydigested heart of 8-week old Sprague-Dawley (SD) rats by densitygradient centrifugation (Transplantation, 57, 1653-1660 (1994)). Rataortic vascular smooth muscle cells (VSMCs) were obtained from 12 weekold SD rats by enzymatic treatment (J. Clin. Invest., 93, 355-360(1994)). These cells were maintained in DMEM medium supplemented with10% (vol/vol) fetal calf serum, penicillin (100 U/ml) and streptomycin(100 μ/ml). The cells were incubated at 37° C. in a humidified 95%air-5% CO₂ atmosphere. The culture medium was routinely changed at 2day-intervals. Both immunopathological and morphological observationrevealed that these cells were endothelial cells and smooth musclecells, respectively.

[0052] Human aortic endothelial cells (five passages) and human VSMCs(five passages) were obtained from Kurabo Co. The endothelial cells wereincubated in a manner similar to the above method in MCDB131 mediumsupplemented with 5% fetal calf serum, epidermal growth factor (10ng/ml), basic fibroblast growth factor (2 ng/ml) and dexamethasone (1μM).

[0053] Endothelial cells in the stationary state were prepared accordingto the method described in J. Clin. Invest., 86, 1690-1697 (1990),ibid., 94, 824-829 (1994).

[0054] (3) Transfection of the HGF Gene into HVJ-Liposomes in vitro

[0055] Endothelial cells or VSMCs were inoculated for sensitization on a6-well plate in a cell count of 10⁶ and proliferated to reach 80%confluence. The cells were washed 3 times with a balanced salt solution(137 mM NaCl, 5.4 mM KCl, 10 mM Tris-HCl, pH 7.6; hereinafterabbreviated as “BSS”) supplemented with 2 mM calcium chloride. To thecells was added 1 ml of a solution of the HVJ-liposome-DNA (containing2.5 mg of lipids and 10 mg of the embedded DNA) obtained in Example 1hereinafter or 1 ml of a solution of the HVJ-liposome-cont obtained inComparative Example 1 hereinafter. The resulting mixture was incubatedat 4° C. for 5 minutes and at 37° C. for a further 30 minutes. The cellswere washed and maintained in a fresh medium containing 10% bovine serumin a CO₂ incubator.

[0056] (4) Assay for the HGF Concentrations in Endothelial Cells andVSMCs

[0057] The concentration of HGF produced from the sensitized endothelialcells and VSMCs was assayed by ELISA. That is, rat or human endothelialcells or VSMCs were inoculated on a 6-well plate (made by Corning) in acell density of 5×10⁴ cells/cm², followed by incubation for 24 hours.The medium was replenished 24 hours after the sensitization, andincubation was continued for a further 48 hours. To investigate if HGFwas released, the sensitized cells (48 hours after sensitization) werewashed and added to 1 ml of a serum-free medium containing 5×10⁻⁷ Minsulin, 5 μg/ml transferrin and 0.2 mM ascorbate. After 24 hours, theculture media was collected, centrifuged at 600 g for 10 minutes andthen stored at −20° C.

[0058] The HGF concentration in the media was determined by an enzymeimmunoassay using an anti-rat HGF antibody or an anti-human HGF antibody(Exp. Cell Res., 210, 326-335 (1994); Jpn. J. Cancer Res., 83, 1262-1266(1992)). A rabbit anti-rat or an anti-human HGF IgG was coated onto a96-well plate (made by Corning) at 4° C. for 15 hours. After blockingwith 3% bovine serum albumin-containing PBS (phosphate buffered saline),the culture medium was added to each well, and incubation was performedat 25° C. for 2 hours. After washing each well 3 times with PBScontaining 0.025% Tween (PBS-Tween), a biotinated rabbit anti-rat HGFIgG or an anti-human HGF IgG was added to each well followed byincubation at 25° C. for 2 hours. After washing with PBS-Tween, eachwell was incubated together with horse radish peroxidase-boundstreptoavidin-biotin complex (PBS-Tween solution). The enzymaticreaction was initiated by adding thereto a substrate solution(containing 2.5 mM o-phenylenediamine, 100 mM sodium phosphate, 50 mMcitrate and 0.015% hydrogen peroxide). The reaction was terminated byadding 1 M sulfuric acid to the system. Absorbance was measured at 490nm. The anti-human HGF antibody is reactive only with human HGF, and notwith rat HGF. The anti-rat HGF antibody is reactive solely with rat HGF,and not with human HGF.

[0059] (5) HGF

[0060] The human and rat recombinant HGFs employed were purified fromthe culture solution of CHO cells or C-127 cells transfected with anexpression plasmid bearing human or rat HGF cDNA (Cell, 77, 261-271(1994); J. Clin. Invest., 93, 355-360 (1994)).

[0061] (6) Statistical Analysis

[0062] All runs were repeated at least 3 times. Data measured are shownby mean±standard error. Statistical analysis of the measured data wasmade according to Duncan's test.

[0063] (7) Hematoxylin-Eosin (HE) Staining and Azan Staining

[0064] Ten days after the gene introduction, the rats having the HGFgene introduced therein were sacrificed by perfusion with heparinizedphysiological saline. Fixation was then made overnight with a 4%paraformaldehyde PBS solution. After fixation, the tissue was embeddedin paraffin. Slides were prepared and stained with HE and Azan in aconventional manner. The slides were examined on a microscope to countthe number of microvessels.

Example 1

[0065] Preparation of HVJ-Liposomes Containing the HGF Expression Vector

[0066] Phosphatidylserine, phosphatidylcholine and cholesterol weremixed with tetrahydrofuran in a weight ratio of 1:4.8:2. By distillingtetrahydrofuran off through a rotary evaporator, the lipid mixture (10mg) was precipitated onto the container wall. After 96 μg of highmobility group (HMG) 1 nuclear protein purified from bovine thymus wasmixed with a BBS (200 μl) solution of plasmid DNA (300 μg) at 20° C. foran hour, the mixture was added to the lipid mixture obtained above. Theresulting liposome-DNA-HMG 1 complex suspension was mixed with a vortex,ultrasonicated for 3 seconds and then agitated for 30 minutes.

[0067] The purified HVJ (strain Z) was inactivated by UV irradiation(110 erg/mm² sec) for 3 minutes immediately before use. BSS was added toand mixed with the liposome suspension (0.5 ml, containing 10 mg of thelipids) obtained above and HVJ (20,000 hemagglutinating units) to makethe total volume 4 ml. The mixture was incubated at 4° C. for 10 minutesand gently agitated at 37° C. for a further 30 minutes. The unreactedHVJ was removed from the HVJ-liposomes by sucrose density gradientcentrifugation. That is, the upper layers in the sucrose densitygradient were collected to give the HVJ-liposomes containing the HGFexpression vector (containing 10 μg/ml of the HGF expression vector).The HVJ-liposomes containing the HGF expression vector is hereinafteroften referred to as HVJ-liposome-DNA.

Example 2

[0068] Administration of the HVJ-Liposome Containing the HGF ExpressionVector to Rats

[0069] The HVJ-liposomes containing the HGF expression vector wereprepared by the method described in the above Example, using 64 μg ofHMG 1 nuclear protein and 200 ìg of plasmid DNA. BSS was added to andmixed with the liposome suspension (0.5 ml, containing 10 mg of thelipids) and HVJ (35,000 hemagglutinating units) to make the whole volume2 ml.

[0070] SD rats (weighing 400-500 g; purchased from Japan Charles River)were anesthetized with intraperitoneal administration of sodiumpentobarbital (0.1 ml/100 mg), warmed and breathing was maintained withan automated breather. The rats were subjected to thoracotomy on theleft side. The HVJ-liposome-DNA or HVJ-liposome-cont (20 μl) wascarefully injected directly through the cardiac apex using a 30 Gsyringe.

Comparative Example 1

[0071] Preparation of HVJ-Liposomes Containing No HGF Expression Vector

[0072] A vector bearing no HGF gene was treated in the same manner asdescribed in Example 1 to prepare the HVJ-liposomes containing no HGFexpression vector. The HGF expression vector-free HVJ-liposomes arehereinafter referred to as HVJ-liposome-cont.

Comparative Example 2

[0073] Preparation of HVJ-Liposomes Containing Human TGF-β ExpressionVector

[0074] HVJ-liposomes containing human TGF-β expression vector wereprepared in a manner similar to that described in Example 1 except forusing a human TGF-β expression vector.

[0075] The HVJ-liposomes containing human TGF-expression vector arehereinafter referred to as HVJ-liposome-DNA (TGF-β).

Test Example 1

[0076] Expression of HGF in Rat Coronary Endothelial Cells Sensitizedwith HVJ-Liposome-DNA

[0077] HVJ-liposome-DNA (concentration of the HGF expression vector inliposomes: 10 μg/ml) was sensitized to rat coronary endothelial cells(cell count: 10⁶). HGF production was determined by ELISA. For control,a similar test was conducted using HVJ-liposome-cont. HGF production wasalso determined on the non-sensitized rat coronary endothelial cells(no-treatment group). The results are shown in FIG. 1 (n=6), wherein“HGF” represents the group of rat coronary endothelial cells sensitizedwith HVJ-liposome-DNA.

[0078] As shown in FIG. 1, the rat coronary endothelial cells sensitizedwith HVJ-liposome-DNA produced and secreted HGF at a high level. On theother hand, HGF production was not substantially observed either in theintact group or in the group of rat coronary endothelial cellssensitized with HVJ-liposome-cont.

[0079] Cell counting in the groups tested revealed that the HGFexpression group showed significantly high cell counts.

Test Example 2

[0080] Effects of the Sensitized HGF Expression Vector on Proliferationof Endothelial Cells

[0081] Human endothelial cells were sensitized with HVJ-liposome-cont.The sensitized cells were incubated in the presence or absence ofexogenously added recombinant human HGF (1, 10 and 100 ng/ml) and thecell growth rate (%) was determined. The results are shown in FIG. 2((line) graph, n=6), wherein “DSF” represents the group of endothelialcells sensitized with HVJ-liposome-cont and “HGF” represents the groupof endothelial cells incubated in the presence of recombinant human HGFin a definite concentration (*: P<0.05, **: P<0.01 for DSF).

[0082] The (line) graph shown in FIG. 2 reveals that the growth ofendothelial cells is promoted by the exogenously added HGF.

[0083] The endothelial cells sensitized with HVJ-liposome-DNA(concentration: 10 μg/ml) were similarly incubated, the increase in thenumber of cells was determined, and the cell growth rate (%) wascalculated. For a control, endothelial cells sensitized withHVJ-liposome-cont were also incubated, the increase in the number ofcells was determined, and the cell growth rate (%) was calculated. Theresults are shown in FIG. 2 (bar, n=6), wherein “DSF” designates thegroup of endothelial cells sensitized with HVJ-liposome-cont and “HGF”designates the group of endothelial cells sensitized withHVJ-liposome-DNA (**: P<0.01 for DSF, #: P<0.05 for HGF, 100 ng/ml).

[0084] As is noted from the bar shown in FIG. 2, the results reveal thatthe cell growth rate of the HVJ-liposome-DNA-sensitized endothelialcells is markedly higher than that of the control group andsignificantly high even when compared to that of cells with exogenouslyadded HGF.

[0085] The aforesaid endothelial cells sensitized with HVJ-liposome-DNAwere incubated in the presence or absence of a rabbit anti-human HGFantibody. The increase in the number of cells was determined, and thecell growth rate was calculated. For a control, the endothelial cellssensitized with HVJ-liposome-cont were incubated, and the increase inthe number of cells was determined in a similar manner in order tocalculate the cell growth rate. The rabbit anti-human HGF antibody (10μg/ml) was purified by the method described in Jpn. J. Cancer Res., 83,1262-1266 (1992). This antibody is capable of neutralizing thebiological activity of 10 ng/ml at a concentration of 10 μg/ml. Theanti-human HGF antibody reacts only with human HGF and not with rat HGF,whereas the anti-rat HGF antibody reacts only with rat HGF and not withhuman HGF. Normal rabbit serum IgG (10 μg/ml) was used for the control.

[0086] The results are shown in FIG. 3 (n=6), wherein “control”designates the group of HVJ-liposome-cont-sensitized endothelial cellsincubated in the presence of IgG control; “HGF” designates the group ofHVJ-liposome-DNA-sensitized endothelial cells incubated in the presenceof IgG control; and “HGFab” designates the group ofHVJ-liposome-DNA-sensitized endothelial cells incubated in the presenceof the rabbit anti-human HGF antibody. The cell growth rate (%) isexpressed in terms of relative % when the growth rate in the controlgroup is made 100 (*: P<0.01 for the control group, #: P<0.05 for HGF).As shown in FIG. 3, the growth of HVJ-liposome-DNA-sensitizedendothelial cells was arrested in the presence of anti-human HGFantibody, and the cell growth rate was thus substantially the same asthat of the control group. These results clearly demonstrate that HGF isthe growth factor of endothelial cells.

Test Example 3

[0087] Effects of the Supernatant of the IncubatedHVJ-Liposome-DNA-Sensitized Rat VSMCs on Rat Coronary Endothelial Cells

[0088] The supernatant from the incubated HVJ-liposome-DNA-sensitizedrat VSMCs was added to the rat coronary endothelial cell culture system(cell count: 10⁵) during the stationary phase. After incubation for 3days, the increase in the number of endothelial cells was determined.For a control, the supernatant of the incubatedHVJ-liposome-cont-sensitized rat VSMCs was added to the rat coronaryendothelial cells in a similar manner to that described above, and theincrease in the number of endothelial cells was determined as describedabove. The results are shown in FIG. 4 (n=6), wherein “control”indicates the group to which was added the supernatant from theincubated HVJ-liposome-cont-sensitized rat VSMCs, and “HGF” representsthe group to which was added the supernatant of the incubatedHVJ-liposome-DNA-sensitized rat VSMCs.

[0089] As shown in FIG. 4, a significant increase in the number ofendothelial cells was observed in the group to which was added thesupernatant of the incubated HVJ-liposome-DNA-sensitized rat VSMCs.

[0090] The concentration of HGF in the culture supernatant of the ratVSMCs sensitized with HVJ-liposome-DNA or HVJ-liposome-cont as describedabove was assayed by ELISA using an anti-human HGF antibody and ananti-rat HGF antibody. The HGF concentration in the culture supernatantof non-sensitized VSMCs was also assayed (no-treatment group).

[0091] The results obtained using the anti-human HGF antibody and theanti-rat HGF antibody are shown in FIGS. 5 and 6, respectively (n=6 inboth tests). In the figures, “control” represents the group to which wasadded the supernatant from the incubated HVJ-liposome-cont-sensitizedrat VSMCs; and “HGF” represents the group to which was added thesupernatant from the incubated HVJ-liposome-DNA-sensitized rat VSMCs.

[0092] As shown in FIG. 5, HGF was detected in the supernatant of theHVJ-liposome-DNA-sensitized rat VSMCs, and the HGF concentration wassignificantly higher than that of the control group.

[0093]FIG. 6 also reveals that rat HGF was further detected in thesupernatant of the HVJ-liposome-DNA-sensitized rat VSMCs, and the HGFconcentration was significantly higher than that of the control group.

[0094] As observed in FIGS. 5 and 6, no HGF was present in an amountdetectable by ELISA, in both the supernatants of the intact group andthe control group.

Test Example 4

[0095] Effects of the Supernatant from the IncubatedHVJ-Liposome-DNA-Sensitized Rat Coronary Endothelial Cells on ratCoronary Endothelial Cells

[0096] The supernatant of the incubated HVJ-liposome-DNA-sensitized ratcoronary endothelial cells was added to the rat coronary endothelialcell culture system (cell count: 10⁵) during the stationary phase. Afterincubation for 3 days, the increase in the number of endothelial cellswas examined. For the control, the endothelial cells were incubated in asimilar manner, using culture supernatant from theHVJ-liposome-cont-sensitized rat coronary endothelial cells, and theincrease in the number of endothelial cells was determined. The resultsare shown in FIG. 7, wherein A, B and C represent, respectively, thegroup to which was added the culture supernatant of theHVJ-liposome-DNA-sensitized rat coronary endothelial cells (n=8), thegroup to which was added the culture supernatant of theHVJ-liposome-cont-sensitized rat coronary endothelial cells (n=8), andthe no-treatment group (n=15).

[0097] As shown in FIG. 7, a significant increase in the number ofendothelial cells was observed in the group to which was added theculture supernatant of the HVJ-liposome-DNA-sensitized rat coronaryendothelial cells, whereas in the control group, the cell count wasalmost the same as that of the no-treatment group (control group:0.117±0.002, group A: 0.148±0.03, P<0.01).

[0098] Next, anti-HGF antibody was added to the culture supernatant ofthe HVJ-liposome-DNA-sensitized rat coronary endothelial cells. Theincrease in the number of endothelial cells was determined as describedabove. The results are shown in FIG. 8 (n=8), wherein A represents thegroup to which was added the culture supernatant of theHVJ-liposome-DNA-sensitized rat coronary endothelial cells; B representsthe group to which was added the culture supernatant of theHVJ-liposome-cont-sensitized rat coronary endothelial cells; Crepresents the group to which was added the culture supernatant of theHVJ-liposome-DNA-sensitized rat coronary endothelial cells and anti-HGFantibody; and D represents the group to which was added the supernatantfrom the incubated HVJ-liposome-DNA-sensitized rat coronary endothelialcells and a control antibody.

[0099] As shown in FIG. 8, A and C, the cell growth promoting activityof the culture supernatant of the HVJ-liposome-DNA-sensitized ratcoronary endothelial cells completely disappeared by adding the anti-HGFantibody thereto. The results reveal that the cell growth promotingactivity of the culture supernatant of the HVJ-liposome-DNA-sensitizedrat coronary endothelial cells is attributable to HGF.

Test Example 5 Effects of HVJ-Liposome-DNA-Sensitized Human VSMCs onHuman Endothelial Cells

[0100] Human VSMCs were inoculated on a cell culture insert(manufactured by Coaster, pore diameter of 0.45 μm), and then grown inDMEM medium supplemented with 10% bovine serum. On the other hand, humanendothelial cells were inoculated on a 6-well plate and maintained inDMEM medium supplemented with 10% bovine serum. When the VSMCs reached80% confluence, the VSMCs were incubated at 4° C. for 5 minutes and at37° C. for 30 minutes together with HVJ-liposome-DNA (DNA content in theliposomes: 10 μg) or with HVJ-liposome-cont. After sensitization, theinsert containing the sensitized VSMCs was added to each well containinghuman endothelial cells in the stationary phase. VSMCs and theendothelial cells were co-incubated for 3 days in DMEM mediumsupplemented with 0.5% bovine serum. Thereafter the cell count wasdetermined with a WST-cell counter kit (manufactured by Wako Co.). Theresults are shown in FIG. 9 (n=6). In the figure, “control” representsthe group co-incubated with the HVJ-liposome-cont-sensitized VSMCs, and“HGF” represents the group co-incubated with theHVJ-liposome-DNA-sensitized VSMCs.

[0101] The results shown in FIG. 9 reveal that human VSMCs sensitizedwith HVJ-liposome-DNA could significantly increase the growth ofnon-sensitized human endothelial cells in the stationary phase.

Test Example 6

[0102] Effects of the HVJ-Liposome-DNA-Sensitized Rat VSMCs on RatCoronary Endothelial Cells

[0103] The HVJ-liposome-DNA-sensitized rat VSMCs (cell count: 10⁶) wereco-incubated for 3 days with rat coronary endothelial cells (cell count:10⁵) in the stationary phase. Thereafter, the increase in the number ofendothelial cells was determined. For the control, endothelial cellswere co-incubated in a similar manner using theHVJ-liposome-cont-sensitized rat VSMCs, and the increase in the numberof endothelial cells was determined. The results are shown in FIG. 10(n=6), wherein “control” represents the rat VSMCs group sensitized withthe HVJ-liposome-DNA, and “HGF” represents the rat VSMCs groupsensitized with HVJ-liposome-cont.

[0104] As shown in FIG. 10, the growth of the endothelial cells wasstimulated by HGF released from the HVJ-liposome-DNA-sensitized ratVSMCs, and an increase in the cell count was observed (control group:0.126±0.006, HGF group: 0.156±0.01, P<0.05).

Test Example 7

[0105] Growth of Rat VSMCs Sensitized with HVJ-Liposome-DNA

[0106] Rat VSMCs sensitized with HVJ-liposome-DNA and rat VSMCssensitized with HVJ-liposome-cont were incubated, respectively, tocompare the increase in the cell counts therebetween. Sensitization withHVJ-liposome-DNA did not affect cell growth at all. The results revealthat HGF has no cell growth promoting effect on VSMCs.

Test Example 8

[0107] Induction of Angiogenesis in Rat Heart Muscle Directly Injectedwith HVJ-Liposome-DNA

[0108] Rat heart muscle directly injected with HVJ-liposome-DNA, ratheart muscle directly injected with HVJ-liposome-cont and ratno-treatment heart muscle were stained with HE and Azan, respectively,and examined on a microscope to count the number of microvessels. Theresults are shown in FIG. 11, wherein “HGF” designates the number ofmicrovessels in rat heart muscle directly injected withHVJ-liposome-DNA, and “control” designates the number of microvessels inrat heart muscle directly injected with HVJ-liposome-cont.

[0109] As is seen from FIG. 11, the number of minute blood vesselssignificantly increased in the rat heart muscle injected withHVJ-liposome-DNA, as compared to that in the rat heart muscle injectedwith HVJ-liposome-cont and the rat no-treatment heart muscle. Theseresults indicate that HGF which is capable of causing endothelial cellsto grow, exhibits angiogenesis activity in vivo.

Test Example 9

[0110] Repair of Articular Cartilage by Directly IntroducingHVJ-Liposome-DNA into the Joint

[0111] Ten week-old Fischer's rats were injured at the femoralintercondylaris through the subcartilage using Kirschner's wires of 1.8mm in diameter. One week after the operation, the HVJ-liposome-DNA (100μl/knee) prepared in Example 1 was introduced directly into the joint.For the control, the HVJ-liposome-cont prepared in Comparative Example 1and HVJ-liposome-DNA (TGF-β) prepared in Comparative Example 2 wereadministered directly into the joint in the same amount. Rats weresacrificed 1, 3 and 4 weeks after introduction of these genes, etc. andthe histology of the repaired sites was observed.

[0112] As shown in FIG. 12, the results indicate that the synthesis ofproteoglycan stained with Toluidine Blue was observed 3 weeks afteradministration of the HVJ-liposome-DNA into the joint, displaying thedevelopment of cartilage-like cells. Furthermore, as shown in FIG. 13, 4weeks after administration of the HVJ-liposome-DNA into the joint, therewas observed a tendency to further extend the area of developingcartilage-like cells, in which the synthesis of proteoglycan wasobserved.

[0113] As shown in FIG. 14, where the HVJ-liposome-DNA (TGF-β) preparedin Comparative Example 2 was injected into the joint, development ofsuch cartilage-like cells was not observed even 4 weeks afteradministration. Furthermore, as shown in FIG. 15, where theHVJ-liposome-cont prepared in Comparative Example 1 was injected intothe joint, development of such cartilage-like cells was not observedeven 4 weeks after administration.

[0114] Industrial Applicability

[0115] The medicament of the present invention provides persistenttherapeutic effects, as compared to HGF itself. Moreover, the medicamentof the present invention may be topically applied to the target organsso that the effects can be selectively exhibited, resulting inminimizing the side effects of HGF.

1.-6. (Cancelled)
 7. A method for treating a cardiovascular disease in asubject for which hepatocyte growth factor (HGF) is effective,comprising administering intracoronarily to the subject an expressionvector containing a HGF gene in a therapeutically effective amount. 8.The method of claim 7, wherein said expression vector is encapsulated ina liposome, the membrane of which may be further fused to attenuatedSendai virus particles.
 9. The method of claim 7, wherein saidcardiovascular disease is restenosis after the percutaneous transluminalcoronary agioplasty (PTCA), myocardial infarction, cardiomyopathy orcardiac insufficiency.